NOREPINEPHRINE BIOSYNTHESIS--ROLE OF ASCORBATE

去甲肾上腺素生物合成——抗坏血酸的作用

• 批准号: 2182932
• 负责人: KANDATEGE WIMALASENA
• 金额: $ 10.35万
• 依托单位: WICHITA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182932
• 关键词: active sites; adenosinetriphosphatase; affinity labeling; animal tissue; ascorbate; chromaffin cells; cytochrome b; dopamine beta monooxygenase; electron probe spectrometry; electron transport; enzyme inhibitors; enzyme mechanism; enzyme substrate; enzyme substrate analog; hydrogen transport; membrane transport proteins; method development; microspectrophotometry; molecular dynamics; neuroendocrine system; neurotransmitter biosynthesis; norepinephrine; nutrition related tag; protein purification; protein reconstitution; vitamin metabolism

The role of ascorbic acid (Asc) in the biosynthesis of both catecholamine neurotransmitters and neuropeptide hormones and thus, in overall neuroendocrine functions has been well recognized. Identification of a complex and intricate ascorbate regenerating system (ARS) in neuroendocrine granules further elaborate the central role of Asc in neuroendocrine functions. Although, the recent efforts have been directed towards the understanding of the functions of Asc/ARS in relation to catecholamine neurotransmitter and neuropeptide hormone biosynthesis, progress of these efforts has been hampered by the complexity and the diversity of the effects of intra- and extra-cellular factors. The work proposed herein is focused on the understanding of the role of Asc/ARS in catecholamine neurotransmitter biogenesis and regulation by systematic investigation of the two major Asc-dependent systems, dopamine beta-monooxygenase (DbM) and cytochrome b561, at the molecular level. In the initial phase of our program, we will develop a sensitive, continuous spectrophotometric assay for DbM and extend it to measure the electron flux into neuroendocrine granules via cyt b561 using adrenal chromaffin granule ghosts as a model system. In the second phase, we will develop various Asc-based chemical probes, i.e. alternate electron donors, competitive and suicide inhibitors, multi-substrates/inhibitors, active site directed inhibitors, and photoaffinity labels to examine the Asc site of DbM in detail. The knowledge gained by these studies will be used and extended to examine the chemistry and the specificity of cyt b561 using purified reconstituted systems and then extend it to more complex cyt b561-mediated ARS in chromaffin granule ghosts. In the third phase, we will use the above assay as well as the Asc-based chemical probes to examine the effect of extra- and intra-cellular factors on norepinephrine biosynthesis in granule ghosts and intact chromaffin granules, in order to understand the molecular mechanisms and interrelations between the catecholamine transporter, membrane ATPase, both soluble and membrane bound DbM and the electron transport protein, cyt b561. The results of these studies will yield information which will be valuable in understanding the crucial role of Asc/ARS, in the dynamics of adrenergic neurosecretory vesicles. In addition, these findings could probably be extended into the peptidergic neurosecretory vesicles, since the peptide processing terminal enzyme, peptidyl amidating monooxygenase (PAM), is also Asc-dependent.

抗坏血酸 (Asc) 在儿茶酚胺生物合成中的作用 神经递质和神经肽激素，因此，总的来说 神经内分泌功能已得到充分认识。 识别一个 神经内分泌中复杂的抗坏血酸再生系统（ARS） 颗粒进一步阐述Asc在神经内分泌中的核心作用 功能。 尽管最近的努力是针对 了解 Asc/ARS 与儿茶酚胺相关的功能 神经递质和神经肽激素的生物合成及其进展 工作的复杂性和多样性阻碍了努力 细胞内和细胞外因素的影响。 本文提出的工作是 重点了解 Asc/ARS 在儿茶酚胺中的作用 通过系统研究神经递质的生物发生和调节 两个主要的 Asc 依赖性系统，多巴胺 β-单加氧酶 (DbM) 和 细胞色素 b561，在分子水平上。 在我们的初始阶段 计划中，我们将开发一种灵敏的连续分光光度测定法 用于 DbM 并将其扩展为测量进入神经内分泌的电子通量 通过 cyt b561 使用肾上腺嗜铬颗粒鬼作为模型的颗粒 系统。 第二阶段，我们将开发各种基于Asc的化学品 探针，即替代电子供体、竞争性和自杀抑制剂， 多底物/抑制剂、活性位点定向抑制剂，以及 光亲和标签可详细检查 DbM 的 Asc 位点。 这 这些研究获得的知识将被使用和扩展以检查 使用纯化的重组细胞色素 b561 的化学和特异性 系统，然后将其扩展到更复杂的 cyt b561 介导的 ARS 嗜铬颗粒鬼影。 在第三阶段，我们将使用上述分析 以及基于 Asc 的化学探针来检查额外的影响 和细胞内因素对颗粒鬼去甲肾上腺素生物合成的影响 和完整的嗜铬颗粒，以了解分子 儿茶酚胺转运蛋白之间的机制和相互关系， 膜 ATP 酶，可溶性和膜结合 DbM 和电子 转运蛋白，细胞色素b561。 这些研究的结果将产生 这些信息对于理解其关键作用非常有价值 Asc/ARS，肾上腺素能神经分泌囊泡的动力学。 在 此外，这些发现可能会扩展到肽能 神经分泌小泡，自肽加工末端酶， 肽基酰胺化单加氧酶 (PAM) 也是 Asc 依赖性的。